Detection of pregnancy by identification of the C peptide of relaxin in body fluids of animals

ABSTRACT

The invention provides a process and products for the detection of the C peptide of relaxin in the body fluids of animals, as a positive indication of pregnancy. The invention may employ monoclonal antibodies generated to various epitopes on the C peptide.

This is a continuation-in-part of application Ser. No. 07/390,626, filedAug. 7, 1989, now U.S. Pat. No. 5,089,419.

The present invention relates generally to processes and productsenabling the detection of pregnancy in animals. More specifically, thepresent invention relates to an assay for pregnancy in an animal whichinvolves the detection and/or the qualitative measurement in body fluidsof the presence of the connecting (or C) peptide of the hormone,relaxin.

BACKGROUND OF THE INVENTION

The peptide hormone, relaxin, is a pregnancy-associated polypeptide,which is present in some analogous form in most mammalian species,including human, porcine, equine, canine, rodent and feline. The relaxinmolecule is processed in the body as a single prepropolypeptidecontaining a signal peptide, followed by a B chain, which is connectedby a connecting peptide (C peptide) to an A chain. During processing thesignal peptide and the C peptide are removed from the relaxin molecule.Thus the biologically active relaxin molecule present in the serum isformed of the A and B chains paired by disulfide bonds into anappropriately folded and active polypeptide.

Immunoreactivity of relaxin requires native conformation [B. E. Kemp etal., Vitamins and Hormones, 41:79-115 (1984)]. When the disulfide bondsof the 6300 dalton di-peptide are reduced, immunoreactivity is lost.Similarly, it is difficult to synthesize an active or immunoreactiverelaxin due to the need to properly align the two interchain and oneintrachain disulfides, even when a portion of the B-chain carboxyterminus is deleted to improve its solubility properties [B. E. Kemp etal., cited above].

The amino acid sequences of the prepropolypeptide of relaxin in porcine,rat and human are known and published in, for example, Bruce E. Kemp andHugh D. Nile, Vitamins and Hormones, Vol. 41, pp. 79-115 (1984). Avariety of mammalian species of relaxin and its prepropolypeptide formsdemonstrate highly conserved genetic regions. The C peptide of relaxinis a larger sequence than the relaxin polypeptide itself. Because the Cpeptide is not itself a hormone, it is not as sensitive to degradationin the excretory or other systems.

Relaxin has been inconsistently reported in some instances as detectablein urine and in other instances as not detectable. See, e.g., WestGerman patent application No. 3236267, published Apr. 5, 1984 whichdiscusses a purification technique for the recovery of relaxin fromurine; and U. Fuchs et al., "Presence of Immunoreactive Relaxin in HumanFemale Urine", Arch. Gynecol. 237 (Suppl.): 383 (1985). Relaxin may bedenatured or degraded before being excreted and, as such, lose itsimmunoreactivity. Without these disulfide bonds it is probable that therelaxin molecule loses its antigenicity, if it is in fact present inurine.

The presence of relaxin in serum is presently the only good earlyindicator of true pregnancy in dogs [B. G. Steinetz et al., Biol.Reproduct., 37:719-725 (1987) (Steinetz I); and B. G. Steinetz et al.,Am. J. Vet. Res., 50:68-71 (1989) (Steinetz II)]. Currently, canineserum relaxin is measured with a radioimmunoassay (RIA) over a three dayprotocol [E. M. O'Byrne et al., Proc. Soc. Exo. Biol. and Med.,152:272-276 (1976); and O. D. Sherwood et al., Endocrinology,96:1106-1113 (1975)]. Pregnancy is indicated upon observation ofdetectable amounts of relaxin, usually observed in pregnant dogs aroundthe twenty-eighth day after the luteinizing hormone (LH) peak (dayzero). The serum relaxin RIA test, however, still requires the help of aveterinarian or trained technician to draw blood samples.

Problems remain with utilizing this assay as a convenient method ofpregnancy detection in animals. For example, serum diagnosis ofpregnancy in animals is simply impractical as a means for theanimals'owner or breeder to routinely practice. There is a need forreliable pregnancy detection methods because certain animals,particularly dogs, often show overt symptoms of pseudopregnancyindistinguishable from the symptoms of actual pregnancy.

Thus there remains a need in the art for convenient means for detectionof the condition of pregnancy in animals, particularly for domesticanimals such as dogs and cats.

SUMMARY OF THE INVENTION

The present invention meets the perceived need in the art by providing amethod for diagnosis of pregnancy in animals, which encompasses thedetection in body fluids of the C peptide of relaxin. The ability todetect the presence of this peptide in body fluids enables rapid,convenient and reliable pregnancy detection. At the present time,however, urine comprises the preferred mode because of the non-invasivenature of collection techniques.

As one aspect, the present invention provides a method for detectingpregnancy in an animal which involves identifying the presence in bodyfluids of the C peptide of relaxin. According to this invention a sampleof a selected body fluid from the animal is contacted with an antibodycapable of binding to an antigenic site on the C peptide of the animal.Preferably this method is applied to detection of pregnancy in domesticanimals, such as dogs and cats. However, this assay may also haveutility in detection of pregnancy in other animals, including, e.g.,horse, cattle.

The antibody for use in the method of this invention may be a polyclonalantibody, polyclonal antisera, or, most preferably, a monoclonalantibody Presently preferred antibodies for use in this assay arecapable of binding both to a site on the C peptide of the tested animaland to a contiguous sequence of rat C peptide, which contains regions ofgenetic conservation with other species C peptide.

In one embodiment of the method of this invention the label associatedwith the antibody is capable of visual detection. Preferable labels mayinclude enzyme systems capable of generating colorimetric signals, suchas horseradish peroxidase (HRP) and tetramethylbenzidine (TMB) oralkaline phosphatase and indoxyl phosphate, or systems generatingvisible agglutinations.

In a further aspect of this invention, the method for detecting Cpeptide in body fluids includes contacting the sample with two or moredifferent labelled antibodies. In this embodiment, each antibody iscapable of binding to a different epitope on the C peptide of the animaltested, and is further capable of binding to the C peptide withoutsterically hindering the binding of the other antibodies.

Preferably the first antibody in this method is conjugated to a firstlabel, the second antibody is conjugated to a second label, and so on.The proximity of these enzymes upon attachment to the C peptide in theassay creates a visually detectable signal However, other detectablelabel systems may also be employed in this embodiment of the invention.

Still a further aspect of the present invention is an antibody capableof binding to a site on the selected animal's C peptide and to acontiguous sequence of rat C peptide, which contains regions of geneticconservation with other species C peptide. Preferably the C peptidesinvolved are those of the canine and feline species. While suchantibodies are preferably monoclonal, polyclonal antibodies and antiserasharing the same characteristics are also included in this invention. Inanother embodiment, these antibodies are associated with detectablelabels.

A further aspect of this invention is a diagnostic kit for the detectionof pregnancy in an animal, preferably a canine or feline, comprising anantibody capable of binding to the C peptide of relaxin present in thebody fluids of the animal The antibody in the kit is conjugated to adetectable label. The specific antibodies described herein may also beparts of such a diagnostic kit, as well as typical buffering, washingand other diagnostic reagents conventional in such kits. Conventionalcomponents such as means for holding the sample, vials and the like arealso included.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of preferred embodimentsof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the detection of the C peptide of relaxinin body fluids as a method for identifying pregnancy in an animal,particularly a domestic animal, such as a dog or cat. This inventionresponds to a need in the field of breeding such animals, as well asprovides a simple test for use by the owner's of animals. The method andproducts provided by this invention have a number of advantages incontrast to present methods for pregnancy detection of animals.

The term "body fluids" includes, but is not limited to, tears, saliva,vaginal secretions, serum, and urine. However, because of nonintrusivemeans of collection and the possibility of increased sensitivity, urineis the presently preferred fluid for use in this invention. Althoughhereafter all references to the selected animal will refer to dogs, forsimplicity, it should be understood that the assays taught by thepresent invention may also be applied to pregnancy detection in cats,and other animals, where desirable.

One of the most obvious advantages of this invention is that, in apreferred embodiment of the present invention, detection of a substancein a body fluid such as urine, tears or saliva as opposed to substanceswhich must be obtained from serum, e.g. the relaxin hormone, are clearlyadvantageous both for at home use by non-veterinarian or non-medicalpersons as well as for clinical use because the collection of urine,tears or saliva does not involve an invasive procedure. Thus the methodand products associated therewith may be easily used by persons havingno special clinical training. The presently available pregnancy testsfor certain animals, particularly dogs and cats, require theintercession of a veterinarian.

Another advantage of the present invention is that practice of thepresent invention may allow detection of the C peptide in the bodyfluids of pregnant dogs earlier than the four week stage at which theintact molecule relaxin is capable of detection in the serum. This isbelieved to be possible because the C peptide is apparently not degradedor digested in urine or in serum. It therefore provides a larger numberof binding sites for the attachment of multiple antibodies. The assaysof this invention, developed to detect the C peptide in body fluids,particularly urine, may thus employ a larger number of antibodiesdirected to different epitopes on the C peptide. This aspect of the Cpeptide thus enables the use of sandwich assays, among others, which arecharacterized by increased sensitivity to detection of the presence of Cpeptide in the urine. It is believed that these advantages are similarlyoffered by other body fluids such as saliva, vaginal secretions, andtears.

Additionally, detection in serum of the C peptide of relaxinspecifically, is believed to offer advantages over prior art methodswhich rely on the detection of the entire relaxin hormone Because thehormone relaxin is known to change physical characteristics withinserum, it has proved difficult to design an accurate, reliable method ofdetection for the hormone. The present invention offers the advantage ofbeing specifically designed to detect a stable peptide and thus providea more accurate assay. Further, because the C peptide is not a hormone,it is not as sensitive to degradation as is the relaxin polypeptideitself.

In a preferred embodiment, the present invention deals primarily with anassay for pregnancy detection which involves identification of thepresence of the C peptide in a sample taken from the body fluids of ananimal.

In an assay of the invention, the fluid sample is contacted with anantibody directed to a antigenic site in the sequence of the C peptideof the dog. Such antibodies may be polyclonal antibodies, such as theillustrative antibody used in Example 1, which is a rabbit, anti-human Cpeptide antibody Polyclonal sera and polyclonal antibodies directed tothe C peptide may be developed by conventional means.

Preferably, however, the assays of this invention will employ monoclonalantibodies (Mabs), which are known to have greater specificity to theirtarget. Hereafter in the discussion of this invention, the term Mabswill be employed for convenience However, it is understood thatpolyclonal antibodies may be substituted therefor in any selected assay.Monoclonal antibodies for use in this invention will be directed toepitopes on the C peptide of the dogs. More desirably such Mabs will beemployed which bind to different sites on the C peptide, so that morethan one type of Mab can be employed in an assay to increase thesensitivity of the assay. Specific Mabs are developed to sequences inthe C peptide which have been shown to be conserved among rat, human andporcine species. These Mabs are described in detail in Example 2 below.Briefly described, these antibodies are capable of binding to rat Cpeptide sequences selected from among peptides characterized bysubstantially the same sequences (or fragments thereof) as follows:

    PHE-ILE-ASN-LYS-ASP-ALA-GLU-PRO-PHE;

    ASN-LEU-SER-GLU-GLU-ARG-LYS-ALA-ALA-LEU-SER;

    ASN-GLN-LEU-GLY-GLU-ALA-GLU-ASP-GLY-GLY-PRO-PRO-GLU-LEU-LYS-TYR;

or

    GLU-LEU-LYS-TYR-LEU-GLY-SER-ASP-ALA-GLN-SER-ARG-LYS-LYS-ARG.

It is understood by one skilled in the art that although the peptidesabove were obtained from the rat relaxin C peptide sequences, othermammalian sequences may also be useful where they contain geneticallyconserved hydrophilic sequences of C peptide analogous to sequences oncanine C peptide, and therefore, desirable as immunogens to generateMabs for use in this invention. However, this invention is not limitedby the specific Mabs described in Example 2. These Mabs are illustrativeonly, and conventional techniques described by Kohler and Milstein andmodifications by others, may be employed by one of skill in the art togenerate Mabs useful in the method and products of this invention. Theonly requirement for selection of the appropriate monoclonal antibodyfor use in the practice of this invention is that, where more than oneantibody is provided, the antibodies must be selected which are capableof binding at a sufficient distance from each other on the C peptide soas to prevent steric hindrance.

In the practice of the method of this invention, a variety of assayformats may be employed which use one or more monoclonal antibodies,polyclonal antibodies and antisera capable of binding to antigenicregions on the C peptide of dogs, including those described in theexamples below.

One assay according to this invention is a homogeneous enzymeimmunoassay employing two Mabs directed against different epitopes onthe canine C peptide. The first Mab is preferably conjugated to a firstenzyme (enzyme 1). A second Mab directed against a different epitope ispreferably conjugated to a second enzyme (enzyme 2) which, in proximityto enzyme 1, is capable of producing a color reaction According to thisembodiment of an assay of this invention, a fluid sample from a femaledog is combined in a test tube or other container with both labeledMabs. A color appears only when the fluid sample contains C peptide,because the enzymes are brought into close proximity due to side-by-sidebinding of their antibody conjugates to the C peptide. The productreleased by enzyme 1 reaction can be utilized immediately by enzyme 2 toconvert the enzyme 2 co-reactant into a colored product capable ofvisualization. If the two enzymes are not in close proximity, e.g., inthe absence of C peptide in the sample, the second reaction does nothave a sufficient concentration of product 1 to proceed in generatingsufficient colored product to be visible.

Another assay format employing the antibodies of this invention isperformed as follows: one antibody directed to a first selected epitopeon the C peptide is conjugated by conventional means to a conventionalsolid matrix, such as latex beads. A second antibody directed to asecond epitope on the C peptide is conjugated again by conventionalmeans to a selected enzyme. According to this assay a sample of fluidfrom a female dog is incubated with the first monoclonal and the latexbeads. The beads are then collected generally by trapping the beads bysize or charge, and separating the beads to which C peptide from thefluid is now attached via the first antibody from the fluid sample. Thesecond monoclonal antibody is then added to the beads and allowed toreact with the C peptide bound to the first monoclonal on the beads.After an appropriate reaction time, generally 2 to 30 minutes, the beadsare washed and a substrate capable of reacting with the enzyme label isadded to the beads. In the presence of the substrate any of the secondenzyme labelled antibody which has bound due to the presence of Cpeptide in the fluid sample will react with the substrate and turn thesample blue, thus providing visual indication and confirmation ofpregnancy. Lack of color indicates that no C peptide was present in thefluid sample and the dog that contributed the sample is not pregnant.

An alternative assay employing two or more monoclonal antibodies todifferent epitopes on the C peptide include a variation of the aboveassay in which the solid phase matrix, e.g. latex beads, are bound to asolid surface and the fluid sample is poured over the beads. This typeof assay eliminates the need for collection of the beads and separationof the fluid. Other than these modifications the remaining steps asdescribed immediately above may be performed.

Still a further variation of a diagnostic assay employing two or moreantibodies of this invention is a latex agglutination assay. Such anassay is known to one of skill in the art and may be briefly describedas follows. Two or more Mabs directed to different epitopes on the Cpeptide are bound to different solid matrices, e.g. different latexbeads. Both beads are together in solution, and the urine sample isadded to the solution. Standard Brownian motion keeps the beads insolution. By controlling the concentration of the beads to the amount offluid, in the presence of C peptide in body fluid sampled, the beadshaving different Mabs will form a lattice network with the C peptide.The resulting agglutination or clumps of Mabs-bound beads bound to twoor more sites on the C peptides are thus capable of visualization in aconventional test tube or on a slide.

Additionally, assays may be performed which only utilize a singlemonoclonal antibody capable of recognizing the native C peptide in bodyfluids. Most preferably a classical competition assay format may beemployed. Alternatively an indirect immunoassay employing one Mab to theC peptide may be employed.

Briefly described, a competition assay is performed as follows: A singleMab to the native C peptide, conjugated by conventional means to a solidmatrix, such as latex beads, is reacted with one milliliter of urine forabout 10 minutes. A drop (approximately 50 μl) of synthetic C peptidefragment conjugated to HRP is added and incubated for an additional 5minutes. The beads are then collected, generally by trapping by size orcharge. The beads are then separated. If native C peptide is present inthe sampled body fluid from a pregnant animal, the native C peptide isnow attached to the beads via the Mab. In the case of a fluid samplefrom a non-pregnant animal which is devoid of native C peptide,synthetic C peptide HRP conjugate is now attached via the Mab specificfor the native C peptide fragment. The collection surface is washed withone ml of wash reagent and 0.2 mls of TMB/urea peroxide is added.

After 5 minutes, 0.2 ml of stop reagent is added. A blue color developsin the absence of the C peptide and, therefore, a positive test remainsclear. This competition assay can also be conducted with the latex-Mabconjugated spotted on a membrane or surface of commercial devices suchas those manufactured by Pall Corporation, Porex, or Bio Rad, or withthe Mab spotted on a dipstick. In these cases, the fluid sample and Cpeptide- HRP conjugate are added in sequence following the same reactiontime protocol. The dipstick incubations can be performed in a test tube.

An exemplary indirect assay employing one Mab is described as follows:100 microliter of urine is passed through nitrocellulose membrane of 3to 5 micron porosity. Any C peptide in the sample binds non-specificallyto the membrane. After 1 minute to allow absorption binding, 0.5 ml ofblocking solution is added and incubated for 2 minutes to block theremaining non-specific binding sites on the membrane. This is followedby 0.5 ml of Mab to C peptide conjugated to HRP and diluted in blockingbuffer. The Mab-HRP conjugate is incubated for 10 minutes. This labelledMab will bind to any C peptide bound to the membrane. The potentialsignal is amplified with goat anti-antibody IgG conjugated with HRP in a1:1000 dilution in the blocking buffer and incubated for 5 minutes. Thisanti-antibody will bind to the bound Mab and thus amplify the label. Themembrane is washed with wash reagent and 0.2 ml of TMB/urea peroxide isadded. After 5 minutes, stop reagent is added. A blue color changeindicates the presence of C peptide in the fluid sample.

These and other assay formats employing one or more Mabs are known toone of skill in the art for diagnosis of many types of conditions.Variations of these described assays may be employed in the method ofthis invention.

Detectable labels for attachment to the antibodies of this invention forthe above described assays may also be easily selected by one skilled inthe art of diagnostic assays. Labels detectable visually are preferredfor use in diagnostic "at-home" kits and even in clinical applicationsdue to the rapidity of the signal and its easy readability. Mostpreferred are labels which provide for colorimetric detection of thepresence of the C peptide. A variety of enzyme systems have beendescribed in the art which will operate appropriately in theabove-described assays. As one example of enzyme 1 in the homogeneousassay, glucose oxidase may be employed with glucose as a substrate.Interaction between glucose and glucose oxidase releases peroxide as aproduct. Enzyme 2 may therefore be horseradish peroxidase, which reactswith peroxide and a hydrogen donor, such as tetramethyl benzidine (TMB),producing an oxidized TMB that is seen as a blue color.

In the assays employing colorimetric enzyme systems, such as HRP oralkaline phosphatase, the reaction should be read within approximately 5to 15 minutes, preferably 10 minutes, to obtain an accurate result. Alonger reaction time can lead to color changes induced by trace amountsof enzyme remaining on the reaction surface. Where desirable, a "stop"solution can be employed to disable the enzyme from further reactionafter the above 5 to 15 minute period. It is known, for example, thatsulfuric acid may be added to stop the reaction of HRP. However, theacid causes a change in color from blue to yellow.

The present invention also provides for novel "stop" solutions, for bothHRP and AP, which may be used in any of the above-described assays andalso included in a kit. A "stop" solution for HRP includes about 20 mMsodium azide, made in 0.25 M sodium acetate to a pH of about 5.0. About0.2 to about 0.5 mls of this solution added to the sample containing HRPafter about 10 to 15 minutes, stops further reaction of HRP, retainingthe blue color or no color in the assay sample indefinitely.

A "stop" solution for AP includes 0.1 M tetrasodium ethylene diaminetetraacetic acid, adjusted to a pH of approximately 7.0 with phosphoricacid. When added to the assay sample following the 5 to 15 minutereaction time, this solution stops further reaction of AP with itssubstrate indoxyl phosphate, also retaining indefinitely the color, orlack thereof, indicating a positive or negative test result.

Other such proximal enzyme systems are known to those of skill in theart, including hexokinase in conjunction with glucose-6-phosphatedehydrogenase which react with ATP, glucose, and NAD+ to yield, amongother products, NADH that is detected as increased absorbance at 340 nmwavelength. The loss of absorbance at 340 nm wavelength by the oxidationof NADH is another indicator of positive results in either allostericactivation using phosphofructokinase in conjunction with phosphoenolpyruvate carboxylase and substrates fructose-6-phosphate and NADH orallosteric inhibition using aspartate aminotransferase in conjunctionwith phosphoenol pyruvate carboxylase and substrates oxalacetate,glutamate and NADH. Also, bioluminescence or chemiluminescence can bedetected using, respectively, NAD oxidoreductase with luciferase andsubstrates NADH and FMN or peroxidase with luminol and substrateperoxide.

Other label systems that may be utilized are detectable by other means,e.g., fluorescent compounds, radioactive compounds or elements orimmunoelectrodes coated with one or several antibodies. These and otherappropriate label systems are known to those of skill in the art.

The methods and Mabs described herein may be efficiently utilized in theassembly of a diagnostic kit, which may be used by pet owners andbreeders. Such a diagnostic kit contains the components necessary topractice one or more of the assays described above for the detection ofthe C peptide of relaxin in dogs. Thus, for homogeneous assays the kitmay contain a first Mab directed to a first epitope on the C peptide,which Mab is associated with a first enzyme, a vial for containing thefluid sample, and a second Mab conjugated to the second enzyme, which inproximity to the first enzyme, produces a visible substrate. Otherconventional components of such diagnostic kits may also be included.

Alternatively, a kit may contain a Mab directed against C peptide boundto a solid surface and associated with a first enzyme, a different Mabassociated with a second enzyme, and a sufficient amount of thesubstrate for the first enzyme, which, when added to the urine and Mabs,provides the reactant for the second enzyme, resulting in the colorchange.

For an agglutination assay kit, the Mabs directed against differentepitopes on the C peptide would be provided bound to the latex beads.

Where the detectable label present in association with the antibody isdesigned for non-visual detection, e.g., for radioimmunoassay, thestandard components necessary for this assay, e.g., controls, standardsand the like are included in the kit.

In the diagnostic kits of this invention, reagents are included whichdefine a clear cut end to the color development step, such as the stopsolutions described above.

The following examples illustrate the practice of the present inventionusing urine as the sample body fluid, including the development ofpresently preferred monoclonal antibodies, and various assay methods.These examples illustrated the invention only, and are not limitingthereof. The assays may also be employed in other body fluids.

EXAMPLE 1 Diagnostic Assay for Pregnancy

According to the present invention the following assay was performed todetect the presence of the C peptide in canine urine. In the performanceof this test the breed of dog employed was a beagle. The dog wasapproximately 8 weeks pregnant. A control dog of the same breed whichwas not pregnant was used to determine the efficacy of this assay.

Approximately 100 microliters of canine urine from each dog was appliedto a nitrocellulose membrane device having a pore size between 3 and 5microns. Commercial devices for such use include those manufactured byPall Corporation and Biorad. If the dog providing the urine sample ispregnant, the C peptide in the canine urine will bind non-specificallyto the nitrocellulose membrane in a fairly rapid manner. Afterapproximately 1 minute to allow the absorption binding, a blockingsolution is applied to saturate any remaining non-specific binding siteson the nitrocellulose membrane. A typical blocking solution for such usemay include, in a volume of 0.5 milliliters, 1% bovine serum albumin(BSA), 10% normal goat serum, 0.1% Tween-20, 0.01% thimerosal, 0.5 MNaCl, 50 mM tris, and additional conventional antibiotics to controlbacterial growth. This blocking solution is desirably buffered to a pHof approximately 7.5 with hydrochloric acid. The goat serum and BSA inthe blocking solution serve to block any remaining non-specific bindingsites on the membrane.

After about 2 minutes following the application of the blockingsolution, an antibody specific for the C peptide may be applied onto thenitrocellulose membrane. In the performance of this particular assay theantibody used was a rabbit anti-human antibody to a 34 amino acidsequence in human C peptide, hereafter called anti-HCP34, in a dilutionof 1:100 in the blocking buffer at a total volume of 0.5 milliliters.This polyclonal antibody was generated to a synthetic sequence of humanC peptide and provided by Dr. G. Bryant-Greenwood, Department of Anatomyand Reproductive Biology, John Burns School of Medicine, the Universityof Hawaii. The reaction between the anti-C peptide antibody and any Cpeptide from the canine urine which is bound to the nitrocellulose isallowed to occur for approximately 10 minutes. During this time theanti-C peptide polyclonal antibody will bind to the C peptide on thenitrocellulose membrane and residual liquids with unbound antibody willdiffuse through the membrane.

The membrane is then washed with approximately 1 ml of buffer describedas above except containing no BSA or goat serum. A goat anti-rabbitantibody, produced by conventional methods known to one of skill in theart, and conjugated to horseradish peroxidase (HRP), approximately1:1000 in dilution in blocking buffer, is applied to nitrocellulosemembrane. If the C peptide is present in the urine sample, it binds tothe nitrocellulose membrane and is present on the membrane attached tothe rabbit anti-C peptide antibody. The HRP-labelled goat anti-rabbitantibody will now bind to the anti-C peptide antibody creating a typicalindirect immunoassay. After 5 minutes the membrane is again washed with1 ml of washing buffer as described above.

Approximately 0.2 ml of tetramethylbenzidene (TMB) in urea peroxide isadded to the nitrocellulose membrane. In the presence of horseradishperoxidase, TMB reacts to produce a blue color easily detected visuallyon the nitrocellulose membrane. This color change on the membraneindicates the presence of C peptide in the urine of the tested canine.

In the assay described above a blue color was revealed for the urine ofthe pregnant canines, while the control canine urine did not changecolor on the nitrocellulose paper.

Twenty dogs were tested in this immunoassay, of which 3 were known to bepregnant. The pregnant dogs tested positive in the assay of thisinvention, and were confirmed pregnant by sonogram tests. These dogssubsequently whelped. Seventeen of the dogs were known non-pregnant, andtested non-pregnant in the above assay of this invention.

It is understood by one skilled in the art that this same assay may beperformed by altering several of the assay parameters, e.g., by adheringthe nitrocellulose membrane to a plastic matrix to create a convenientdipstick pregnancy test product. Additionally the amount of the reagentsor samples can be changed, and a different detectable label system maybe employed.

EXAMPLE 2 Generation of C Peptide Epitopes and Monoclonal AntibodiesThereto

In generating Mabs desirable for use in the present invention, thefollowing amino acid sequences were selected and generated syntheticallyby conventional means for use as immunogens in conventional methods forgenerating Mabs. These sequences were selected from areas of geneticconservation among rat, human and porcine C peptide sequences [Kemp etal., cited above].

Peptides were synthesized which corresponded to amino acid residues Nos.77-85 of the rat relaxin C peptide (numbering of amino acids correspondsto the numbering of the rat peptide in Protein Identification Resource,National Biomedical Research Foundation, Georgetown University MedicalCenter, 3900 Reservoir Road, N.W., Washington, D.C. 20007):

    PHE-ILE-ASN-LYS-ASP-ALA-GLU-PRO-PHE.

The entire peptide fragment was synthesized, as well as smaller andslightly larger fragments, including peptides having an additionalcysteine residue on the amino or carboxy terminus of the selectedpeptide.

Another sequence from rat relaxin C peptide selected for generation ofMabs spanned amino acid residues Nos. 94 through 104 and fragmentsthereof:

    ASN-LEU-SER-GLU-GLU-ARG-LYS-ALA-ALA-LEU-SER.

Fragments of this entire sequence were also generated, including anumber of fragments with a cysteine at either the carboxy or aminotermini.

Still another sequence obtained from the rat relaxin C peptide spannedfrom amino acid residue No. 136-151:

    ASN-GLN-LEU-GLY-GLU-ALA-GLU-ASP-GLY-GLY-PRO-PRO-GLU-LEU-LYS-TYR.

Fragments of this entire sequence, including fragments from amino acidresidue Nos. 138-145, optionally containing a cysteine added to thecarboxy or amino termini, were also generated.

Finally for the purposes of this example another sequence of rat relaxinC peptide from amino acid residue No. 148 through the final amino acidresidue No. 162 were generated:

    GLU-LEU-LYS-TYR-LEU-GLY-SER-ASP-ALA-GLN-SER-ARG-LYS-LYS-ARG.

Smaller and larger fragments of this sequence were also generated,optionally containing a carboxy or amino terminal cysteine.

The above-described peptides or contiguous portions thereof weregenerated by solid phase synthesis using a polyamide resin. See, e.g.the method of Merrifield as reported in the Journal of American ChemicalSociety, 85:2149-2154 (1963), involving the stepwise addition ofprotected amino acids to a growing peptide chain which was bound bycovalent bonds to a solid resin particle Reagents and byproducts areremoved from this solid phase synthesis by filtration. The generalconcept of this method depends on attachment of the first amino acid ofthe chain to a solid polymer by a covalent bond and the addition ofsucceeding amino acids one at a time in a stepwise manner until thedesired sequence is assembled.

The amino acids may be attached to any suitable polymer which merely hasto be insoluble in the solvents used and have a stable physical formpermitting ready filtration. The resin or matrix must also contain afunctional group to which the first protected amino acid can be firmlylinked by a covalent bond. Various polymers are suitable for thispurpose including polyamide resins, polystyrene andpolymethylmethacrylate. See, also, for example, Bodansky et al., PeptideSynthesis, Interscience, second edition (1976). As described below, thepeptide may optionally be removed from the solid support and protectinggroups removed.

The cysteines which are optionally attached at the carboxy or aminotermini of the selected fragment provide a sulfhydryl group which may beused to link the selected peptide to a carrier protein for antibodygeneration or alternatively to an enzyme or other detectable label. Inthe generation of antibodies it is well known that small peptides becomemore antigenic when attached to a carrier such as a latex, keyholelimpet hemocyanin or bovine thyroglobulin.

The Mabs useful in the present invention are desirably generated instandard laboratory mice using the now traditional Kohler and Milsteintechniques as well as other modifications thereof known to one of skillin the art for the generation of Mabs. Desirably, mice are injected withthe selected peptide coupled to the solid phase polyamide resin orattached to a carrier protein. The mice may be sequentially boosted withthe same peptide attached to different carriers to stimulate the cellsto recognize the selected peptide as the target.

In the generation of Mabs for use in the following examples, twomonoclonal antibody regimens are followed. In one regimen, the mice areboosted every other day for approximately 3 weeks to obtain Mabsdirected to the selected peptide. Alternatively, to obtain higheraffinity Mabs the mice are boosted approximately every two weeks forbetween 3 to 6 months to generate antibodies selected to the peptidespresented.

The Mabs generated according to this example are capable of bindingselectively to epitopes on the C peptide of canines felines, which arehomologous to the conserved region in the rat relaxin C peptide andhuman relaxin C peptides. This battery of Mabs directed to the selectedepitopes are useful in identifying and binding to epitopes on the canineor feline C peptides for use in assays employing one, or desirably,multiple antibody binding sites to enhance the detection of the label.

For use in these assays the label is desirably attached to the Mab byconventional methods known to one of skill in the art. For example, apresently preferred method to attach an enzyme label (horseradishperoxidase or alkaline phosphatase) to the Mab uses theheterobiofunctional conjugation agent, sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, (SMCC). 4 mg of Mab areequilibrated in 0.1 M sodium phosphate buffer, pH 6.0 by chromatographythrough an excellulose column (manufactured by Pierce). Fractionscontaining the Mab are concentrated to 0.45 ml in a centricon 10(manufactured by Amicon) by centrifugal concentration. 50 μl of 0.1 M2-mercaptoethylamine in 0.1 M sodium phosphate buffer, pH 6.0 are addedand incubated for 1.5 hours at 37° C. The reduced Mab is thenequilibrated in 0.1 M sodium phosphate, 5 mM EDTA, pH 6.0 throughexcellulose chromatography and concentrated to 0.5 ml in a centricon 10.4 mg of horseradish peroxidase are equilibrated in 50 mM sodium borate,pH 7.6 by chromatography through an excellulose column. Fractionscontaining the horseradish peroxidase are concentrated to 0.5 ml bycentrifugal concentration in a centricon 10. 1 mg of SMCC is added andincubated for 30 to 60 minutes at 30° C. The SMCC labelled horseradishperoxidase is then equilibrated in 0.1 M Tris buffer, pH 7.0 throughexcellulose chromatography and concentrated to 1 ml using a centricon10. The SMCC labelled horseradish peroxidase is then combined with thereduced Mab and incubated for 20 hours at 4° C. 10 μl of 0.1 M2-mercaptoethylamine in 0.1 M sodium phosphate, pH 6.0 is added andincubated for 20 minutes at room temperature. The Mab horseradishperoxidase conjugate is fractionated through Sephadex G-200chromatography in 0.15 M sodium chloride, 10 mM Tris, 0.01% thimerosal,pH 7.5 and stored at 4° C.

Other conjugation methods include one- or two-step glutaraldehydemethod, periodate-oxidation method, water soluble carbodiimide method,homobifunctional imidoesters, hydroxysuccinimide esters, or maleimidemethods, other heterobifunctional hydroxysuccinimidyl ester, maleimide,pyridyl disulfide or active halogen reaction methods, photoreactivephenyl azide methods, and avidin-biotin methods.

EXAMPLE 3 Detection of C Peptide in Vaginal Fluid

Dipsticks, coated with C peptide monoclonal antibody, 5E10, wereprepared in the following manner: Streptavidin in 0.1 MCarbonate-Bicarbonate buffer, pH 9.5, was coated on polystyrenedipsticks for 90 minutes at 37°. Dipsticks were then incubated for 90minutes at 37° in biotinylated goat-anti-mouse IgG in 0.1 M phosphatebuffered saline, pH 7.4 (PBS). The streptavidin-biotinylatedgoat-anti-mouse IgG bridge was then crosslinked with dimethylpimelimidate (DMP) in 0.2 M ethanolamine, pH 8.3. Next, monoclonalantibody, 5E10, was added in PBS, then crosslinked with DMP. Thedipsticks are then blocked with 1% (w/v) bovine serum albumin, 5% (w/v)sucrose in PBS, and air dried.

Synthetic C peptide containing a terminal cysteinyl residue wasconjugated to horseradish peroxidase (HRP) using SMCC. TMB/urea peroxidewas used as the color development substrate.

The following steps were performed to assay for the presence of Cpeptide: The 5E10 coated dipsticks were inserted 3 inches towards theanterior vaginal region of pregnant and non-pregnant beagles, and heldin place for 5 minutes. The dipsticks were then incubated for 5 minutesin 1 ml of HRP conjugated synthetic C peptide. Next, the dipsticks wererinsed for 10 seconds under a gently stream of room temperature tapwater, excess water was removed by vigorous shaking, then colordevelopment proceeded for 10 minutes in 1 ml of TMB/urea peroxide. Colordevelopment was stopped by removing the dipsticks and adding 0.2 ml of20 mM sodium azide in 0.2 sodium acetate, pH 5.0.

C peptide in the vaginal fluids of pregnant bitches saturated monoclonalantibody 5E10 binding sites. This was observed as clear colordevelopment tubes in contrast to the distinct blue results fromnon-pregnant bitches.

It is understood by one of skill in the art, however, that the presentinvention is not limited to the specific Mabs described by the examplesabove, since other Mabs may be easily generated by one of skill in theart according to the teachings of the present invention.

Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. For example, use of otherappropriate Mabs and detectable labels are contemplated in theperformance of this invention. Such modifications and alterations to thecompositions and processes of the present invention are believed to beencompassed in the scope of the claims appended hereto.

What is claimed is:
 1. A method for non-invasive detection of pregnancyin a mammal comprising identifying the presence of the C peptide ofrelaxin in a sample of body fluids selected from the group consisting ofsaliva, tears and vaginal secretions, said method comprising the stepsof (a) contacting the sample with at least one antibody specific for anantigenic site on the C peptide; (b) incubating the mixture for asufficient period of time and in appropriate conditions to allowcomplexing of the antibody and antigen in the sample; (c) detecting anycomplex; and (d) correlating any complex to the presence or absence ofpregnancy.
 2. The method according to claim 1 wherein said mammal isselected from the group consisting of canines and felines.
 3. The methodaccording to claim 1 wherein said antibody bears a detectable label. 4.The method according to claim 3 wherein said antibody is specific for acontiguous amino acid sequence containing region of genetic conservationwith the C peptide of other mammalian species.
 5. The method accordingto claim 4 wherein mammalian species is rat, and said contiguous saidsequence of C peptide is selected from the group consisting of

    PHE-ILE-ASN-LYS-ASP-ALA-GLU-PRO-PHE;

    ASN-LEU-SER-GLU-GLU-ARG-LYS-ALA-ALA-LEU-SER;

    ASN-GLN-LEU-GLY-GLU-ALA-GLU-ASP-GLY-GLY-PRO-PRO-GLU-LEU-GLY-TYR;

and

    GLU-LEU-LYS-TYR-LEU-GLY-SER-ASP-ALA-GLN-SER-ARG-LYS-LYS-ARG.


6. The method according to claim 3 wherein said antibody is selectedfrom the group consisting of polyclonal antibodies, polyclonal antisera,and a monoclonal antibody.
 7. The method according to claim 3 whereinsaid label is capable of visual detection.
 8. The method according toclaim 7 wherein said label comprises an enzyme system capable ofgenerating colorimetric signals.
 9. The method according to claim 7wherein said enzyme system is selected from the group consisting ofhorseradish peroxidase and TMB or alkaline phosphatase and indoxylphosphate
 10. The method according to claim 6 wherein said label isselected from among the groups consisting of fluorescent compounds,radioactive compounds or elements.
 11. A method according to claim 1wherein step (a) comprises contacting said fluid sample with two or moredifferent labelled antibodies, each antibody specific for a differentepitope on the C peptide of said mammal without sterically hindering thebinding of the other antibodies.
 12. The method according to claim 11wherein the first antibody is conjugated to a first label, said secondantibody is conjugated to a second label, and the proximity of the firstlabel to the second label upon attachment to the C peptide creates avisually detectable signal.
 13. The method according to claim 1 whereinstep (a) comprises exposing a fluid sample to an antibody specific forsaid native C peptide, which antibody is bound to a solid matrix andthen exposing the sample to a synthetic C peptide associated with alabel, followed by washing to separate any unbound material, wherein thepresence of C peptide in said body fluid is indicated by the binding thenative C peptide to the bound antibody providing no detectable label,while the absence of C peptide in said fluid is indicated by the bindingof the labelled synthetic C peptide to the bound antibody which resultsin the appearance of the detectable label.
 14. The method according toclaim 1 further comprising incubating the mixture step (b) with alabelled synthetic C peptide capable of complexing with the antibody inthe absence of native C peptide wherein the detection of the complexbetween antibody and labelled synthetic C peptide indicates the absenceof pregnancy in said mammal.